Marker-Free Plant Genome Editing via Guide Polynucleotide/Cas
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for genetic modification in plants often result in random and unpredictable integration of transgenes, and there is a need for more efficient and effective methods to modify specific genomic locations without incorporating selectable transgene markers.
Innovation Solution
A guide polynucleotide/Cas endonuclease system is employed to modify or alter target sites in the plant genome, using a guide polynucleotide and Cas endonuclease to introduce double-strand breaks at specific locations, allowing for the integration of polynucleotides of interest without a selectable transgene marker.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional transgene integration methods (Agrobacterium infection or biolistic particle bombardment) are used, then foreign DNA sequences can be inserted into the plant genome, but the integration occurs in a random and unpredictable copy number
Solution Approach 1:
The transgene construct is divided into two separate T-DNA regions: one containing the desired transgene and another containing the selectable marker. These segments are integrated at different genomic locations through two separate Agrobacterium transformation events, allowing precise control over integration sites while maintaining the ability to select for successful transformants
Solution Approach 2:
A selectable marker gene serves as an intermediary element that facilitates the identification and selection of plants that have successfully integrated the transgene. The marker is integrated at a different genomic location than the transgene itself, acting as a separate selectable trait that enables screening without being physically linked to the transgene integration site
2Manufacturing precision
If site-specific recombination is used to target a predetermined sequence, then transgene integration can be controlled, but a selectable marker must be included as part of the introduced DNA sequences
Solution Approach 1:
The selectable marker is separated from the transgene into a different T-DNA region, allowing the transgene to be integrated without the marker. This segmentation enables the marker to be used for selection purposes while the transgene remains as a clean, marker-free integration at the target site
Solution Approach 2:
The selectable marker is extracted from the transgene construct and placed in a separate integration event. This allows the marker to be removed from the final transgene product, resulting in a cleaner genetic modification that does not contain the selectable marker sequence
3Manufacturing precision
If multiple transformation events are used to separate transgene and marker integration, then the transgene can be integrated without the selectable marker, but the process becomes more complex
Solution Approach 1:
The selectable marker serves multiple functions: it enables selection of transformants, facilitates identification of successful integration events, and can be used for breeding purposes. By placing it in a separate T-DNA region, it maintains its utility while allowing the transgene to be integrated cleanly without the marker sequence
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This system enables precise genetic modifications in plants by introducing targeted double-strand breaks, facilitating the integration of desired nucleotides without the need for exogenous selectable markers, thereby enhancing the efficiency and specificity of genetic editing.
Implementation Method 1
One method for inserting or modifying a DNA sequence involves homologous DNA recombination by introducing a transgenic DNA sequence flanked by sequences homologous to the genomic target
Implementation Method 2
artificially induced site-specific genomic double-stranded breaks in plant cells were repaired by homologous recombination with exogenously supplied DNA
Data Source
AI summary
Compositions and methods are provided for genome modification of a target sequence in the genome of a plant or plant cell, without incorporating a selectable transgene marker. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed, without incorporating a selectable transgene marker. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Breeding methods and methods for selecting plants utilizing a guide polynucleotide/Cas endonuclease system are also disclosed. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell, without incorporating a selectable transgene marker.